6DG4

Structure of the Chaetomium thermophilum Ulp1-like SUMO protease catalytic domain

6DG4 の概要

• エントリーDOI: 10.2210/pdb6dg4/pdb
• 分子名称: Ulp1-like SUMO protease, SULFATE ION, GLYCEROL, ... (4 entities in total)
• 機能のキーワード: sumo hydrolase, ubiquitin-like protease, smt3 hydrolase, desumoylating enzyme, cysteine protease, sumo processing enzyme, smt3 processing enzyme, hydrolase
• 由来する生物種: Chaetomium thermophilum
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 30010.11

構造登録者

Lima, C.D.,Baytshtok, V. (登録日: 2018-05-16, 公開日: 2018-07-11, 最終更新日: 2023-10-11)

主引用文献

Lau, Y.K.,Baytshtok, V.,Howard, T.A.,Fiala, B.M.,Johnson, J.M.,Carter, L.P.,Baker, D.,Lima, C.D.,Bahl, C.D.
Discovery and engineering of enhanced SUMO protease enzymes.
J. Biol. Chem., 293:13224-13233, 2018

PubMed Abstract: Small ubiquitin-like modifier (SUMO) is commonly used as a protein fusion domain to facilitate expression and purification of recombinant proteins, and a SUMO-specific protease is then used to remove SUMO from these proteins. Although this protease is highly specific, its limited solubility and stability hamper its utility as an reagent. Here, we report improved SUMO protease enzymes obtained via two approaches. First, we developed a computational method and used it to re-engineer WT Ulp1 from to improve protein solubility. Second, we discovered an improved SUMO protease via genomic mining of the thermophilic fungus , as proteins from thermophilic organisms are commonly employed as reagent enzymes. Following expression in , we found that these re-engineered enzymes can be more thermostable and up to 12 times more soluble, all while retaining WT-or-better levels of SUMO protease activity. The computational method we developed to design solubility-enhancing substitutions is based on the RosettaScripts application for the macromolecular modeling suite Rosetta, and it is broadly applicable for the improvement of solution properties of other proteins. Moreover, we determined the X-ray crystal structure of a SUMO protease from to 1.44 Å resolution. This structure revealed that this enzyme exhibits structural and functional conservation with the SUMO protease, despite exhibiting only 28% sequence identity. In summary, by re-engineering the Ulp1 protease and discovering a SUMO protease from , we have obtained proteases that are more soluble, more thermostable, and more efficient than the current commercially available Ulp1 enzyme.

PubMed: 29976752
DOI: 10.1074/jbc.RA118.004146

実験手法

X-RAY DIFFRACTION (1.442 Å)